US20230295312A1 - Antibody against human il-4ra and use thereof - Google Patents

Antibody against human il-4ra and use thereof Download PDF

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US20230295312A1
US20230295312A1 US17/419,109 US201917419109A US2023295312A1 US 20230295312 A1 US20230295312 A1 US 20230295312A1 US 201917419109 A US201917419109 A US 201917419109A US 2023295312 A1 US2023295312 A1 US 2023295312A1
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amino acid
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Baiyong Li
Yu Xia
Zhongmin Wang
Peng Zhang
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07—ORGANIC CHEMISTRY
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
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    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7155—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders

Definitions

  • the present invention relates to the field of allergic disease treatment and molecular immunology, and particularly, to an anti-human interleukin-4-receptor A antibody, a pharmaceutical composition or a kit comprising the same, and use thereof.
  • Interleukin-4-receptor is a transmembrane receptor that exists in two distinct forms: type I IL-4R, which consists of an IL-4R a subunit (referred to as IL-4RA herein) with high affinity and a ⁇ c subunit with moderate affinity, and binds to interleukin-4 (IL-4) and mediates IL-4 induced cell proliferation, activation and other biological functions; and type II IL-4R, which consists of an IL-4R a subunit (IL-4RA) with high affinity and an interleukin-13-receptor a subunit (IL-13Ra), and is a homologous functional receptor for interleukin-13 (IL-13) capable of binding to IL-13 (Wang and Secombes, Cytokine, 2015, 75(1):8-13).
  • type I IL-4R which consists of an IL-4R a subunit (referred to as IL-4RA herein) with high affinity and a ⁇ c subunit with moderate affinity, and binds
  • IL-4R is expressed in T cells, B cells, hematopoietic stem cells and endothelial cells, epithelial cells, muscle, fibroblasts, hepatocytes, and brain tissue.
  • IL-4RA along with ⁇ c subunit or IL-13R a subunit, activates various non-receptor protein tyrosine kinases in cytoplasm after IL-4 binds to its receptor, further initiating downstream signal transduction pathways (Nelms et al., Annu. Rev. Immunol. , 1999, 17:701-738; LaPorte et al., Cell, 2008, 132(2):259-272).
  • IL-4RA can bind to IL-4 and IL-13 with high affinity, and is a major functional subunit of the foregoing type I and type II IL-4R.
  • the inhibition of IL-4RA can effectively block relevant biological functions mediated by IL-4 and IL-13 (Gessner et al., Immunobiology , 2000, 201:285).
  • IL-4 is a pleiotropic cytokine secreted by immune cells such as CD4 + T cell subsets, B cells, mast cells and the like; IL-13 is produced mainly by activated T cells (Th2 in mice) in human, and has various biological effects on monocytes (macrophages), B lymphocytes, NK cells, vascular endothelial cells and the like.
  • IL-4 and IL-13 can exert corresponding effector functions in a variety of cells, such as T cells, B cells, eosinophils, mast cells, basophils, airway smooth muscle cells, respiratory tract epithelial cells, fibroblasts and endothelial cells, which are the major effector cells responsible for the development of allergic diseases such as allergic rhinitis and food allergy, and asthma (May et al., Cytokine , 2015, 75(1):89-116); furthermore, airway smooth muscle cells, respiratory tract epithelial cells, fibroblasts and endothelial cells, etc.
  • T cells such as T cells, B cells, eosinophils, mast cells, basophils, airway smooth muscle cells, respiratory tract epithelial cells, fibroblasts and endothelial cells, which are the major effector cells responsible for the development of allergic diseases such as allergic rhinitis and food allergy, and asthma (May et al., Cytokine , 2015, 75(1):89-
  • T and B cell dysfunctions are the major mechanisms of autoimmune diseases
  • IL-4 is involved in the development of autoimmune diseases by activating downstream signaling pathways through binding to IL-4R on the surface of immune cells (May et al., Cytokine , 2015, 75(1):89-116).
  • IL-4/IL-13 pathways play an important role in asthma pathology (Chatila et al., Trends in Molecular Med. , 2004, 10(10):493-499) as well as in other diseases outlined elsewhere herein. Airway hyperreactivity, mucus0 hypersecretion, and airway remodeling, etc. are the key pathological features of asthma. Studies have demonstrated that IL-4 and IL-13 are involved in the development and maintenance of the above pathological processes (March et al., Cytokine , 2015, 75(1):89-116).
  • IL-13 is considered as a key cytokine for triggering airway hyperreactivity (AHR), and IL-4 is a major cause of Th2 immune cell polarization and IgE production (Wynn et al., Annu. Rev. Immunol. , 2003, 21:425-456).
  • Atopic dermatitis is a serious chronic inflammatory skin disease, mainly characterized by severe itching, pronounced eczematous changes and dry skin.
  • Atopic dermatitis usually starts in infants, and continues for a lifetime in some patients. It may seriously affect the quality of life of the patients due to chronic recurrent eczematous rash, severe itching, sleep deficit, dietary restrictions, and psychosocial reasons.
  • IL-4/IL-13 are considered as a key driver of the persistent intrinsic inflammation of atopic dermatitis (Malajian et al., Cytokine , 2015, 73(2):311-318).
  • dupilumab an anti-human IL-4RA monoclonal antibody
  • FDA U.S. Food and Drug Administration
  • IL-4, IL-13 and IL-4R pathways are involved in the development and progression of chronic obstructive pulmonary disease, rhinosinusitis and tumors, and the inhibition of IL-4RA has the potential for treating chronic obstructive pulmonary disease (Jin Lin et al., Journal of Practical Medicine , 2014, 30(22):3543-3544), rhinosinusitis, pulmonary fibrosis and tumors (May et al., Cytokine , 2015, 75(1):89-116; Guo Changkuo et al., Chemistry of Life , 2017, 37(3):413-418).
  • anti-human IL-4RA monoclonal antibody dupilumab in combination with glucocorticoids can significantly ameliorate symptoms and reduce nasal polyps (Bachert et al., JAMA , 2016, 315(5):469-79).
  • the anti-human IL-4RA antibody medicament has wide application prospect, and can be used for treating allergic diseases, such as allergic rhinitis, asthma, allergy and atopic dermatitis, while treating rhinosinusitis, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis, tumor, autoimmune diseases and the like. Therefore, the development of antibody medicaments with high affinity to human IL-4RA for treating allergic diseases with better efficacy and less severe toxic and side effects poses great significance. However, available anti-human IL-4RA antibody medicaments showed insufficient affinities, and there’s still a need for an anti-human IL-4RA antibody with high affinity.
  • the inventors used mammalian cell expression systems to express recombinant IL-4RA as an antigen to immunize mice, and obtained hybridoma cells by fusion of mouse spleen cells and myeloma cells. The inventors obtained the following hybridoma cell lines by screening a large number of the samples.
  • the hybridoma cell line 13E5 is capable of secreting and producing a specific antibody (named as 13E5) that specifically binds to the human IL-4RA, and the antibody can block the binding of human IL-4RA to IL-4 effectively.
  • the inventors have creatively prepared humanized anti-human IL-4RA antibodies (named as 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L4 and 13E5 H4L2, respectively).
  • the hybridoma cell line 18H7 is capable of secreting and producing a specific antibody (named as 18H7) that specifically binds to the human IL-4RA, and the antibody can block the binding of human IL-4RA to IL-4 effectively.
  • the inventors have creatively prepared humanized anti-human IL-4RA antibodies (named as 18H7 H1L1, 18H7 H2L2, 18H7 H2L3 and 18H7 H3L2, respectively).
  • the hybridoma cell line 20G10 is capable of secreting and producing a specific antibody (named as 20G10) that specifically binds to the human IL-4RA, and the antibody can block the binding of human IL-4RA and IL-4 effectively.
  • the inventors have creatively prepared a humanized anti-human IL-4RA antibody (named as 20G10 H3L3).
  • the foregoing antibodies can effectively bind to the human IL-4RA, block the binding of human IL-4RA to the ligand IL-4 or IL-13 thereof, and inhibit the activation of the downstream signaling pathway of the human IL-4 RA.
  • the antibodies have potential in preparing a medicament for preventing and treating allergic rhinitis, asthma, allergy, atopic dermatitis, rhinosinusitis, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis and autoimmune diseases.
  • One aspect of the present invention relates to an antibody or an antigen-binding fragment thereof, the antibody comprising:
  • the antibody comprises:
  • amino acid sequences of the CDR regions of the antibodies in (1) to (13) above are analyzed by technical means well known to those skilled in the art, for example, by a VBASE2 database.
  • the antibodies 13E5, 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L4 and 13E5 H4L2 disclosed herein share the same HCDR1-3 and LCDR1-3.
  • amino acid sequences of the 3 HCDR regions of the heavy chain variable region are as follows:
  • HCDR1 GYTFTEYT SEQ ID NO: 9
  • amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
  • the antibodies 18H7, 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, and 18H7 H3L2 disclosed herein share the same HCDR1-3 and LCDR1-3.
  • amino acid sequences of the 3 HCDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFTFSSSY SEQ ID NO: 15
  • amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
  • CDR2 SAS SEQ ID NO: 19
  • the antibody 20G10 H3L3 disclosed herein has the following HCDR1-3 and LCDR1-3.
  • amino acid sequences of the 3 HCDR regions of the heavy chain variable region are as follows:
  • HCDR1 GFSLSTSGMG SEQ ID NO: 130
  • HCDR2 IWWADDK SEQ ID NO: 131
  • HCDR3 ARITRGNSAMDF SEQ ID NO: 132
  • amino acid sequences of the 3 CDR regions of the light chain variable region are as follows:
  • LCDR1 ENVYSY SEQ ID NO: 133
  • LCDR2 NAK SEQ ID NO: 134
  • LCDR3 QHHYGIPWT SEQ ID NO: 135
  • the antibody further comprises framework regions (FRs) in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 49, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 49; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 9
  • FR-H1
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 53, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 53; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 9
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 57, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 57; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 61, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 61; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 62, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 65, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 65; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 66, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 69, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 69; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 70, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 9
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 73, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 73; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 74, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 77, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 77; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 78, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 81, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 81, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 81; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 82, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set froth in SEQ ID NO: 85, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 85, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 85; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 9
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 89, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 89, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 89; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 90, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 9
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 93, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 93, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 93; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 94, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 97, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 97; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 98, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 101, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 101, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 101; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 102, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 105, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 105, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 105; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 106, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 109, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 109, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 109; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 110, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 9
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 113, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 113, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 113; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 114, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 117, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 117, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 117; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 118, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the heavy chain variable region, preferably the FRs including FR-H1, FR-H2, FR-H3 and FR-H4, wherein the FR-H1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 136, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 136, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 136; the FR-H2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 137, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%,
  • the antibody further comprises FRs in the light chain variable region, preferably the FRs including FR-L1, FR-L2, FR-L3 and FR-L4, wherein the FR-L1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 140, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO: 140, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the amino acid sequence set forth in SEQ ID NO: 140; the FR-L2 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 141, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%,
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 9, 10 and 11, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 12, 13 and 14.
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 15, 16 and 17, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 18, 19 and 20.
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 12, 13 and 14, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 9, 10 and 11.
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 18, 19 and 20, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 15, 16 and 17.
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 130, 131 and 132, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 133, 134 and 135.
  • One aspect of the present invention relates to an isolated polypeptide comprising the sequences set forth in SEQ ID NOs: 133, 134 and 135, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 130, 131 and 132.
  • One aspect of the present invention relates to an isolated polypeptide comprising a sequence selected from the sequences set forth in SEQ ID NOs:
  • One aspect of the present invention relates to an isolated polypeptide comprising a sequence selected from the sequences set forth in SEQ ID NOs:
  • the antigen-binding fragment is selected from Fab, Fab′, F(ab′) 2 , Fd, Fv, dAb, Fab/c, complementarity determining region (CDR) fragment, single chain antibody (e.g., scFv), bivalent antibody and domain antibody.
  • the antibody is a humanized antibody, a chimeric antibody or a multispecific antibody (e.g., bispecific antibody).
  • the antibody binds to human IL-4RA protein with a K D less than about 10 -5 M, e.g., less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.
  • the K D is measured by a Fortebio molecular interaction instrument.
  • the antibody binds to human IL-4RA protein with an EC 50 less than about 100 nM, e.g., less than about 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less.
  • the EC 50 is measured by indirect ELISA.
  • the antibody comprises a constant region
  • the constant region is derived from a species other than murine, e.g., from a human antibody, preferably from a human IgG, more preferably from IgG4.
  • the constant region of the antibody is humanized, e.g., the heavy chain constant regions are Ig gamma-4 chain C regions, preferably the Ig gamma-4 chain C region of GenBank ACCESSION No: P01861.1; the light chain constant regions are Ig kappa chain C regions, preferably the Ig kappa chain C region of GenBank ACCESSION No: P01834.
  • Another aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 9, 10 and 11, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 12, 13 and 14.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 15, 16 and 17, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 18, 19 and 20.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 12, 13 and 14, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 9, 10 and 11.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 18, 19 and 20, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 15, 16 and 17.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 130, 131 and 132, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 133, 134 and 135.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising the sequences set forth in SEQ ID NOs: 133, 134 and 135, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising the sequences set forth in SEQ ID NOs: 130, 131 and 132.
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising a sequence selected from the sequences set forth in SEQ ID NOs: 2, 6, 22, 26, 30, 34 and 38, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising a sequence respectively selected from the sequences set forth in SEQ ID NOs: 4, 8, 24, 28, 32, 36 and 40, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • One aspect of the present invention relates to an isolated polynucleotide encoding a polypeptide comprising a sequence selected from the sequences set forth in SEQ ID NOs: 4, 8, 24, 28, 32, 36 and 40, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences, or an amino acid sequence having one or more (preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences, wherein the polypeptide specifically binds to human IL-4RA as part of an anti-human IL-4RA antibody, the antibody further comprising a sequence respectively selected from the sequences set forth in SEQ ID NOs: 2, 6, 22, 26, 30, 34 and 38, a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 9
  • the polynucleotide molecule comprises or consists of the sequences set forth in SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41, SEQ ID NO: 45 or SEQ ID NO: 126, or a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences.
  • the polynucleotide molecule comprises or consists of the sequences set forth in SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 47 or SEQ ID NO: 128, or a sequence having at least 80%, 85% or 90%, preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequences.
  • Yet another aspect of the present invention relates to a vector comprising any one of the polynucleotide molecules disclosed herein.
  • Yet another aspect of the present invention relates to a host cell comprising any one of the polynucleotide molecules disclosed herein, or the vector disclosed herein.
  • Yet another aspect of the present invention relates to a method for preparing any one of the antibodies or the antigen-binding fragments thereof disclosed herein, comprising culturing the host cell disclosed herein in a suitable condition, and isolating the antibody or the antigen-binding fragment thereof from the cell cultures.
  • One aspect of the present invention further provides an antibody conjugate comprising the anti-human IL-4RA antibody or the antigen-binding fragment thereof, and a conjugated moiety coupled thereto, wherein the conjugated moiety is a purification tag (e.g., a His tag), a cytotoxic agent or a detectable label.
  • the conjugated moiety is a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.
  • One aspect of the present invention further provides a multispecific antibody, preferably a bispecific antibody, comprising the anti-human IL-4RA antibody or the antigen-binding fragment thereof, and an antibody or an antigen-binding fragment against another antigen and/or another antigenic epitope.
  • a multispecific antibody preferably a bispecific antibody, comprising the anti-human IL-4RA antibody or the antigen-binding fragment thereof, and an antibody or an antigen-binding fragment against another antigen and/or another antigenic epitope.
  • One aspect the present invention further provides a fusion protein comprising any one of the anti-human IL-4RA antibodies or the antigen-binding fragments thereof disclosed herein.
  • One aspect the present invention further provides a kit comprising any one of the antibodies or the antigen-binding fragments thereof disclosed herein, or the antibody conjugate or the multispecific antibody disclosed herein.
  • the kit further comprises a second antibody that specifically recognizes the antibody or the antigen-binding fragment thereof; optionally, the second antibody further comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.
  • a detectable label such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.
  • Yet another aspect of the present invention relates to use of any one of the antibodies or the antigen-binding fragments thereof disclosed herein, or the antibody conjugate or the multispecific antibody disclosed herein in preparing a kit for detecting the presence or level of human IL-4RA in a sample or for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of an allergic disease, a tumor, an autoimmune disease, a skin infection, tissue fibrosis, rhinosinusitis, nasal polyps and chronic obstructive pulmonary disease, wherein preferably the allergic disease is selected from atopic dermatitis, allergic rhinitis, asthma and allergy, and more preferably, atopic dermatitis includes moderate and severe atopic dermatitis.
  • Yet another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising any one of the antibodies or the antigen-binding fragments thereof disclosed herein or the antibody conjugate, the multispecific antibody or the fusion protein disclosed herein, and optionally, a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition is used alone or in combination with one or more medicaments.
  • the pharmaceutical composition and the combined medicament are separately present in the kit.
  • Yet another aspect of the present invention relates to use of any one of the antibodies or the antigen binding fragments thereof disclosed herein or the antibody conjugate, the multispecific antibody or the fusion protein disclosed herein in preparing:
  • One aspect of the present invention relates to use of any one of the antibodies or the antigen binding fragments thereof disclosed herein or the antibody conjugate, the multispecific antibody or the fusion protein disclosed herein in preparing a medicament for treating a disease selected from: an atopic dermatitis including moderate and severe atopic dermatitis; nasal polyps; asthma; a skin infection; an autoimmune disease, and the like.
  • Yet another aspect of the present invention relates to an in vivo or in vitro method comprising administering a cell comprising the antibody or the antigen-binding fragment thereof disclosed herein, the antibody conjugate, the multispecific antibody or the fusion protein disclosed herein, or administering to a subject in need an effective amount of any one of the antibodies or the antigen-binding fragments thereof or the antibody conjugate, the multispecific antibody or the fusion protein disclosed herein.
  • the method is selected from:
  • the in vitro method is non-therapeutic and/or non-diagnostic.
  • Yet another aspect of the present invention relates to use of any one of the antibodies or the antigen-binding fragments thereof or the antibody conjugate or the multispecific antibody disclosed herein in preparing a medicament for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of an allergic disease, a tumor, a skin infection, an autoimmune disease, tissue fibrosis, rhinosinusitis, nasal polyps and chronic obstructive pulmonary disease.
  • the allergic disease is selected from atopic dermatitis, allergic rhinitis, asthma and allergy.
  • the medicament is in a dosage form suitable for oral administration to the gastrointestinal (GI) tract.
  • the dosage form is selected from tablets, capsules, pills, powders, granules, emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the medicament is in a form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.
  • Yet another aspect of the present invention relates to a method for the prevention and/or treatment and/or adjuvant treatment and/or diagnosis of an allergic disease, a tumor, an autoimmune disease, tissue fibrosis, a skin infection, rhinosinusitis, nasal polyps, chronic obstructive pulmonary disease, comprising administering any of the antibody or the antigen-binding fragment thereof, the antibody conjugate, or the multispecific antibody disclosed herein to a subject in need.
  • the allergic disease is selected from atopic dermatitis, allergic rhinitis, asthma and allergy.
  • the term “antigen-binding fragment” means a protein or portion of a protein that specifically binds to a given antigen.
  • a portion of an antibody comprising amino acid residues that interact with an antigen and confer the antibody the specificity and affinity for the antigen is referred to as an “antigen-binding fragment”.
  • the antigen-binding fragment generally comprises one or more complementary-determining regions (CDRs).
  • CDRs complementary-determining regions
  • Certain antigen-binding fragments further comprise one or more framework regions (FRs).
  • CDRs are amino acid sequences that contribute to antigen binding specificity and affinity.
  • an antibody refers to an intact immunoglobulin of any isotype or an antigen-binding fragment thereof that can compete with an intact antibody for specifically binding to a target antigen, and includes, for example, chimeric, humanized, fully human, and bispecific antibodies or antigen-binding fragments thereof.
  • Such “antibodies” are antigen-binding proteins.
  • An intact antibody generally comprises at least two full-length heavy chains and two full-length light chains, but, in some cases, may comprise fewer chains, such as an antibody naturally occurring in Camelidae species that may comprise only a heavy chain.
  • an antibody or antigen-binding fragment thereof may be derived from a single source only, or may be “chimeric”, i.e., different portions of an antibody may be derived from two different sources as further described below.
  • Antibodies or antigen-binding fragments thereof can be produced in hybridomas by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies.
  • the term “antigen-binding fragment” (or simply “fragment”) of an “antibody” or “immunoglobulin chain” (heavy or light chain) includes a portion of an antibody (whether obtained or synthesized) that lacks at least some of the amino acid residues present in the full length of the antibody but is capable of specifically binding the antigen.
  • Such fragments are biologically active as they specifically bind to a target antigen and can compete with other antibodies or antigen binding fragments thereof for specifically binding to a given epitope.
  • such fragments will retain at least one CDR present in the full-length light or heavy chain of the antibody, and in some embodiments, will comprise a single heavy and/or light chain or a portion thereof.
  • Such biologically active fragments can be produced by recombinant DNA techniques, or, for example, by enzymatic or chemical cleavage of intact antibodies.
  • Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab′, F(ab′) 2 , Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including, but not limited to, human, mouse, rat, Camelidae species, and rabbit.
  • a functional portion of an antibody disclosed herein can be covalently bound to a second protein or a small molecule to generate a therapeutic agent that targets a particular target in the body, thereby having bifunctional therapeutic properties or having an extended serum half-life, such as a fusion protein.
  • antibody full-length chain As used herein, the terms “antibody full-length chain”, “full-length antibody”, “intact antibody” and “whole antibody” are used interchangeably herein to refer to such an antibody having a substantially similar structure to a natural antibody structure or having a heavy chain that comprises an Fc region as defined herein.
  • the term “light chain” includes full-length light chains and fragments thereof having a sufficient variable region sequence to confer the binding specificity.
  • the full-length light chain comprises a variable region domain VL and a constant region domain CL.
  • the variable region domain of the light chain is at the amino terminus of the polypeptide.
  • Light chains include kappa ( ⁇ ) and lambda ( ⁇ ) chains.
  • the term “heavy chain” includes full-length heavy chains and fragments thereof having a sufficient variable region sequence to confer the binding specificity.
  • the full-length heavy chain comprises a variable region domain VH and 3 constant region domains CH1, CH2 and CH3.
  • the VH domain is at the amino terminus of the polypeptide and the CH domains are at the carboxyl terminus, in which CH3 is closest to the carboxyl terminus of the polypeptide.
  • the heavy chain may be of any isotype, including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.
  • Fab fragment consists of one light chain, CH1 and the variable region of one heavy chain.
  • the heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.
  • Fc region comprises two heavy chain fragments comprising the CH1 and CH2 domains of an antibody.
  • the two heavy chain fragments are held together by two or more disulfide bonds and the hydrophobic interaction of the CH3 domains.
  • Fab′ fragment comprises portions of one light chain and one heavy chain including the VH domain and the CH1 domain and the region between the CH1 and CH2 domains) such that interchain disulfide bonds can be formed between the two heavy chains of two Fab′ fragments to give an F(ab′) 2 molecule.
  • F(ab′) 2 fragment comprises two light chains and two heavy chains containing portions of the constant region between the CH1 and CH2 domains such that interchain disulfide bonds are formed between the two heavy chains.
  • the F(ab′) 2 fragment consists of two Fab′ fragments held together by disulfide bonds between the two heavy chains.
  • Fv region comprises the variable regions from the heavy and light chains, but lacks the constant regions.
  • Fd fragment refers to an antibody fragment consisting of VH and CH1 domains (Ward et al., Nature , 341:544-546 (1989)).
  • dAb fragment consists of a VH domain (Ward et al., Nature 341:544-546 (1989)).
  • Fab′-SH′′ is the designation herein for Fab′, wherein one or more cysteine residues of the constant domain carry a free thiol group.
  • Fab/c fragment is an intermediate formed by pepsin digestion of an immunoglobulin, which combines the advantages of Fab and Fc regions, i.e., strong diffusibility and low metabolic clearance in vivo, while retaining high affinity (Liu Jianjun, Chinese Journal of Cellular and Molecular Immunology , 1989(4):29-29).
  • single-chain antibody is an Fv molecule in which the heavy and light chain variable regions are connected by a flexible linker to form a single polypeptide chain (which forms an antigen binding region) (see, e.g., Bird et al., Science, 242:423-426 (1988), and Huston et al., Proc. Natl. Acad. Sci. , USA, 90:5879-5883 (1988)).
  • Single-chain antibodies are described in detail in International Patent Publication No. WO88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference.
  • domain antibody is an immunofunctional immunoglobulin fragment that comprises only the variable region of the heavy chain or the light chain.
  • two or more VH regions are covalently linked by a peptide linker to generate a multivalent domain antibody (particularly a bivalent domain antibody).
  • the two VH regions of the bivalent domain antibody may target the same or different antigens.
  • bivalent antigen-binding protein or “bivalent antibody” comprises two antigen-binding sites. In some cases, the two binding sites have specificity against the same antigen.
  • the bivalent antibody may be bispecific.
  • multispecific antigen-binding protein or “multispecific antibody” is an antigen-binding protein or antibody that targets more than one antigen or epitope.
  • bispecific As used herein, the term “bispecific”, “dual-specificity” or “bifunctional” antigen-binding protein or antibody is a hybrid antigen-binding protein or antibody having two different antigen-binding sites, respectively.
  • a bispecific antibody is a multispecific antigen-binding protein or multispecific antibody, and can be produced by a variety of methods, including but not limited to, fusion of hybridomas or linkage of Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. , 79:315-321; Kostelny et al., 1992, J. Immunol. , 148:1547-1553.
  • the two binding sites of a bispecific antigen-binding protein or antibody will bind to two different epitopes that are present in the same or different protein targets.
  • the terms “mAb” and “monoclonal antibody” refer to an antibody or a fragment of an antibody that is derived from a group of highly homologous antibodies, i.e., from a group of identical antibody molecules, except for natural mutations that may occur spontaneously.
  • the monoclonal antibody has a high specificity for a single epitope on an antigen.
  • the polyclonal antibody, relative to the monoclonal antibody generally comprises at least two or more different antibodies which generally recognize different epitopes on an antigen.
  • Monoclonal antibodies can generally be obtained using hybridoma technique first reported by Kohler et al. ( Nature , 256:495, 1975), but can also be obtained using recombinant DNA technique (see, e.g., U.S. Pat. No. 4,816,567).
  • humanized antibody refers to an antibody or antibody fragment obtained when all or a part of CDR regions of a human immunoglobulin (receptor antibody) are replaced by the CDR regions of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody having expected specificity, affinity or reactivity.
  • donor antibody may be a non-human (e.g., mouse, rat or rabbit) antibody having expected specificity, affinity or reactivity.
  • some amino acid residues in the framework regions (FRs) of the receptor antibody can also be replaced by the amino acid residues of corresponding non-human antibodies or by the amino acid residues of other antibodies to further improve or optimize the performance of the antibody.
  • epitope refers to a site on an antigen at which an immunoglobulin or antibody specifically binds. “Epitope” is also called in the field as an “antigenic determinant”.
  • the epitope or antigenic determinant generally consists of chemically active surface groups of molecules such as amino acids, carbohydrates or sugar side chains, and usually has specific three-dimensional structural characteristics and specific charge characteristics.
  • the epitope generally includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-consecutive amino acids in a unique spatial conformation, which can be “linear” or “conformational”. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E.
  • polypeptide or “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the term is also used to refer to an amino acid polymer in which one or more amino acid residues are analogs or mimetics of naturally existing amino acids, and for naturally existing amino acid polymers.
  • the term may also include, for example, amino acid polymers that have been modified by addition of saccharide residues to form glycoproteins, or have been phosphorylated.
  • Polypeptides and proteins can be produced by naturally existing cells and non-recombinant cells; or they may be produced by genetically engineered or recombinant cells, and comprise a molecule having the amino acid sequence of a native protein or a molecule having deletions, insertions and/or substitutions in one or more amino acids of the native sequence.
  • polypeptide and “protein” include antibodies, such as anti-human IL-4RA antibodies (also referred to as IL-4RA antibodies), IL-4RA-binding proteins, antibodies or sequences having deletions, insertions, and/or substitutions in one or more amino acids of an antigen-binding protein.
  • IL-4RA antibodies also referred to as IL-4RA antibodies
  • IL-4RA-binding proteins antibodies or sequences having deletions, insertions, and/or substitutions in one or more amino acids of an antigen-binding protein.
  • polypeptide fragment refers to a polypeptide having amino-terminal deletions, carboxyl-terminal deletions, and/or internal deletions as compared to a full-length protein. Such fragments may also contain modified amino acids as compared to the full-length protein. In certain embodiments, such fragments are about 5 to 500 amino acids in length. For example, a fragment can be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400 or 450 amino acids in length.
  • Useful polypeptide fragments include immunologically functional fragments of antibodies, including binding domains. In the case of human IL-4RA antibodies, useful fragments include but are not limited to, CDR regions, variable domains of heavy or light chains, portions of antibody chains, variable domains just comprising 2 CDRs, or the like.
  • human IL-4RA refers to its mature peptide (Genbank ID: NP_001244336.1).
  • IL-4 and IL-13 are the main endogenous agonists of IL-4RA.
  • IL-4RA refers to human IL-4RA.
  • a “derivative” of a polypeptide is a polypeptide (e.g., an antigen-binding protein or antibody) that is chemically modified in other manners than insertion, deletion or substitution, e.g., by conjugation with another chemical moiety, e.g., a PEG-conjugated polypeptide.
  • isolated refers to “obtained by artificial means from a natural state”. If a certain “isolated” substance or component appears in nature, it may be that change occurs in its natural environment, or that it is isolated from the natural environment, or both. For example, a certain non-isolated polynucleotide or polypeptide naturally exists in a certain living animal, and the same polynucleotide or polypeptide with a high purity isolated from such a natural state is called isolated polynucleotide or polypeptide.
  • isolated does not exclude the existence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector allows for the expression of the protein encoded by the inserted polynucleotide
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic substance elements carried by the vector can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phages or M13 phages; and animal viruses.
  • artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC)
  • phages such as lambda phages or M13 phages
  • animal viruses include but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as lambda phages or M13 phag
  • Animal viruses that can be used as vectors include, but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as lentiviruses
  • adeno-associated viruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • papovaviruses such as SV40.
  • a vector can contain a variety of elements that control expression, including, but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term “host cell” refers to cells that can be introduced with vectors, including, but not limited to, prokaryotic cells such as E. coli or bacillus subtilis , fungal cells such as yeast cells or aspergillus , insect cells such as S2 drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.
  • prokaryotic cells such as E. coli or bacillus subtilis
  • fungal cells such as yeast cells or aspergillus
  • insect cells such as S2 drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.
  • an antibody that specifically binds to an antigen refers to that the antibody binds to the antigen with an affinity (K D ) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less.
  • K D refers to a dissociation equilibrium constant for a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen.
  • the K D value is the dissociation equilibrium constant.
  • K D k dis /k on .
  • a smaller equilibrium dissociation constant indicates a more intensive antibody-antigen binding and a higher affinity between the antibody and the antigen.
  • k on (association rate constant) is the rate of antigen-antibody complex formation, and a smaller k on suggests a faster binding of an antibody to an antigen.
  • k dis (dissociation rate constant) is the rate at which an antibody dissociates from an antigen-antibody complex, and a smaller k dis suggests a slower rate for the antibody dissociating from the antigen and a firmer binding between the antibody and the antigen.
  • an antibody binds to an antigen (e.g., L1 protein) with a dissociation equilibrium constant (K D ) of less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less, for example, as determined on a BIACORE surface plasmon resonance (SPR) instrument or a ForteBio molecular interaction instrument.
  • K D dissociation equilibrium constant
  • the terms “monoclonal antibody” and “McAb” have the same meaning and can be used interchangeably; the terms “polyclonal antibody” and “PcAb” have the same meaning and can be used interchangeably; the terms “polypeptide” and “protein” have the same meaning and can be used interchangeably.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • hybridoma and “hybridoma cells” can be used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cells”, subclones and progeny cells of the hybridoma are also included.
  • percent sequence identity As used herein, the terms “percent sequence identity” and “percent sequence homology” are used interchangeably.
  • sequence similarity refers to the correlation of the sequences of two or more protein or polypeptide molecules, as determined by aligning and comparing the sequences.
  • Percent identity refers to the percentage of identical amino acid residues in the molecules compared, and can be calculated based on the size of the smallest molecule for comparison. For such calculations, gaps in the alignment (if any) must be addressed by a particular mathematical model or computer program (i.e., an “algorithm”).
  • substantially identical when used for polypeptides, refers to that two peptide sequences, when optimally aligned, for example using the programs GAP or BESTFIT, using default gap weights provided by the programs, have at least 70%, 75% or 80% sequence identity, at least 90% or 95% sequence identity, or at least 97%, 98% or 99% sequence identity.
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • Constant amino acid substitution is one in which the amino acid residue is replaced with another amino acid residue having a side chain R group that possesses similar chemical properties (e.g., charge or hydrophilicity). Generally, conservative amino acid substitutions will substantially retain the functions and properties of the protein.
  • the percent sequence identity may be elevated to correct for the conservative nature of the substitution.
  • Methods for making this adjustment are well known to those skilled in the art. See, e.g., Pearson, Methods Mol. Biol. , 243:307-31 (1994).
  • Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic hydroxy side chain: glycine, alanine, valine, leucine and isoleucine, 2) aliphatic hydroxy side chain: serine and threonine, 3) amide-containing side chain: asparagine and glutamine, 4) aromatic side chain: phenylalanine, tyrosine and tryptophan, 5) basic side chain: lysine, arginine and histidine, 6) acidic side chain: aspartic acid and glutamic acid, and 7) sulfur-containing side chain: cysteine and methionine.
  • the conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine
  • a conservative substitution is any change with a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al., Science, 256:1443-45 (1992), which is incorporated herein by reference.
  • a “moderately conservative” substitution is any change with a non-negative value in the PAM250 log-likelihood matrix.
  • Sequence identity of polypeptides is usually measured by sequence analysis software. Protein analysis software matches sequences using a measure of similarity assigned to different substitutions, deletions and other modifications (including conservative amino acid substitutions). For example, GCG, including programs such as “Gap” and “Bestfit” which (using default parameters specified by the program) can be used to determine sequence homology or sequence identity between closely related polypeptides (e.g., homologous polypeptides from different biological species) or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1 (University of Wisconsin, WI). Polypeptide sequences can also be compared using FASTA with default or recommended parameters.
  • FASTA e.g., FASTA2 and FASTA3 which provides alignments for regions of optimal overlap between the challenge and query sequences and percent sequence identities
  • FASTA2 and FASTA3 Another preferred algorithm when comparing sequences to a database containing massive sequences from different organisms is the computer program BLAST, in particular, blastp or tblastn (using default parameters provided by the program).
  • BLAST Altschul et al., Mol. Biol., 215:403-410 (1990); Al tschul et al., Nucleic Acids Res., 25:3389-402 (1997).
  • the present invention has the following advantages:
  • the anti-human IL-4RA antibody disclosed herein can bind to human IL-4RA with high affinity, and inhibit relevant cellular biological effects mediated by human IL-4 and human IL-13, such as cell proliferation, IL-4 and IL-13 induced CD23 expression level up-regulation and the like, by blocking the binding of human IL-4 and human IL-13 to human IL-4RA.
  • the antibody has advantages of high activity, exclusion of species difference and the like, and can be used in preparing medicaments for blocking the binding of human IL-4 and IL-13 to human IL-4RA and in preparing medicaments for treating or preventing allergic diseases such as allergic rhinitis, asthma, allergy and atopic dermatitis (including moderate and severe atopic dermatitis), sinusitis, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis, and autoimmune diseases, thus having prospect for application and marketing.
  • allergic diseases such as allergic rhinitis, asthma, allergy and atopic dermatitis (including moderate and severe atopic dermatitis), sinusitis, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis, and autoimmune diseases, thus having prospect for application and marketing.
  • FIG. 1 Binding activity of 13E5 and dupilumab to antigen IL4RA-mFc.
  • FIG. 2 Binding activity of 13E5 H1L1, 13E5 H2L2, 13E5 H3L3 and dupilumab to antigen IL4RA-mFc.
  • FIG. 3 Binding activity of 13E5 H4L2, 13E5 H4L4 and dupilumab to antigen IL4RA-mFc.
  • FIG. 4 Activity of 13E5 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 5 Activity of 13E5 H1L1, 13E5 H2L2, 13E5 H3L3 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 6 Activity of 13E5 H4L2, 13E5 H4L4 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 7 Binding activity of 18H7 and dupilumab to antigen IL4RA-mFc.
  • FIG. 8 Binding activity of 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, 18H7 H3L2, 18H7 H3L3 and dupilumab to antigen IL4RA-mFc.
  • FIG. 9 Activity assay of 18H7 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 10 Activity assay of 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, 18H7 H3L2 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 11 Activity assay of 20G10 H3L3, 13E5 H4L4 and dupilumab in competing with human IL4-N-His for binding to human IL4RA-mFc.
  • FIG. 12 Affinity constant assay of 13E5 H4L4 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 13 Affinity constant assay of 18H7 H1L1 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 14 Affinity constant assay of dupilumab to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 15 Affinity constant assay of 13E5 H1L1 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 16 Affinity constant assay of 13E5 H2L2 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 17 Affinity constant assay of 13E5 H3L3 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM and 0.39 nM, respectively.
  • FIG. 18 Affinity constant assay of 13E5 H4L2 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 19 Affinity constant assay of 18H7 H2L2 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 20 Affinity constant assay of 18H7 H3L2 to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 25 nM, 12.5 nM, 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 21 Affinity constant assay of dupilumab to human IL4RA.
  • the antibody concentrations for the curve pairs from top to bottom were 6.25 nM, 3.13 nM, 1.56 nM, 0.78 nM and 0.39 nM, respectively.
  • FIG. 22 13E5 H1L1, 13E5 H2L2 and dupilumab inhibiting IL-4-induced TF-1 cell proliferation.
  • FIG. 23 13E5 H1L1, 13E5 H2L2 and dupilumab inhibiting IL-13-induced TF-1 cell proliferation.
  • FIG. 24 13E5 H4L2, 13E5 H4L4 and dupilumab inhibiting IL-4-induced TF-1 cell proliferation.
  • FIG. 25 13E5 H4L2, 13E5 H4L4 and dupilumab inhibiting IL-13-induced TF-1 cell proliferation.
  • FIG. 26 18H7 H1L1, 18H7 H2L2 and dupilumab inhibiting IL-4-induced TF-1 cell proliferation.
  • FIG. 27 18H7 H1L1, 18H7 H2L2 and dupilumab inhibiting IL-13-induced TF-1 cell proliferation.
  • FIG. 28 18H7 H1L1, 13E5 H4L4 and dupilumab inhibiting IL-4-induced CD23 expression up-regulation in monocytes.
  • FIG. 29 18H7 H1L1, 13E5 H4L4 and dupilumab inhibiting IL-13-induced CD23 expression up-regulation in monocytes.
  • FIG. 30 13E5 H4L4 inhibiting increase of epidermal thickness in B-hIL4Ra mouse skin inflammation model.
  • FIG. 31 Pathological sections showing epidermal thickness in mice of all experimental groups (HE staining, 400 ⁇ ).
  • a. control normal saline
  • b. dupilumab 80 mg/kg
  • d. hIgG4 80 mg/kg
  • e. 13E5 H4L4 80 mg/kg
  • BALB/C mice were purchased from Guangdong Medical Laboratory Animal Center.
  • dupilumab the reference antibody dupilumab VAB 16F3-1 (hereinafter referred to as dupilumab) was produced by Akeso, Inc., the sequence of which can be found in Patent Application No. PCT/US2007/021210 granted to Regeneron Pharmaceuticals, Inc.
  • the heavy chain variable region of antibody VAB 16F3-1 is set forth in SEQ ID NO: 124, with the constant region being an Ig gamma-1 chain C region, ACCESSION No. P01857, while the coding sequence of the light chain variable region of antibody VAB 16F3-1 is set forth in SEQ ID NO: 125, with the constant region being an Ig kappa chain C region, ACCESSION No. P01834).
  • the antigen IL-4RA-mFc for producing anti-IL-4RA antibody was a fusion protein of human IL-4RA mature peptide (Genbank ID: NP_001244336.1) and mFc tag (SEQ ID NO: 121) synthesized by Akeso, Inc., and was used for immunizing BALB/C mice (purchased from Guangdong Medical Laboratory Animal Center). Spleen cells of immunized BALB/C mice (purchased from Guangdong center for medicine laboratory animals) and mouse myeloma cells were fused to form hybridoma cells with reference to existing cell fusion techniques (e.g., Stewart, S.J., “Monoclonal Antibody Production”, in Basic Methods in Antibody Production and Characterization, Eds.
  • IL 4RA-hFc protein IL-4RA is described above, and hFc is a human IgG Fc purification tag, specifically Ig gamma-1 chain C region, Genbank ID: P01857, positions 114-330
  • IL-4RA is described above
  • hFc is a human IgG Fc purification tag, specifically Ig gamma-1 chain C region, Genbank ID: P01857, positions 114-330
  • hybridoma cell lines obtained by indirect ELISA screening were subjected to competitive ELISA to select hybridoma cell lines secreting monoclonal antibodies that compete with ligand IL4-N-his (IL4 NCBI Gene ID: AAH70123.1) for binding to IL 4RA-hFc.
  • Two hybridoma cell lines stably secreting anti-human IL-4RA antibodies were obtained by limiting dilution.
  • the hybridoma cell lines were named as LT018, LT008 and LT009, and the secreted monoclonal antibodies were named as 20G10, 13E5 and 18H7.
  • Hybridoma cell line LT018 (IL4RA-20G10) was deposited at China Center for Type Culture Collection (CCTCC) on Dec. 25, 2019 with an accession number CCTCC NO: C202010, and a preservation address of Wuhan University, Wuhan, China, postal code: 430072.
  • Hybridoma cell line LT008 (IL4RA-13E5) was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 21, 2018 with an accession number CCTCC NO: C2018131, and a preservation address of Wuhan University, Wuhan, China, postal code: 430072.
  • Hybridoma cell line LT009 (IL4RA-18H7) was deposited at China Center for Type Culture Collection (CCTCC) on Jun. 21, 2018 with an accession number CCTCC NO: C2018132, and a preservation address of Wuhan University, Wuhan, China, postal code: 430072.
  • the cell lines LT018, LT008 and LT009 prepared as described above were separately cultured with a chemical defined medium (CD medium; containing 1% streptomycin) in a 5% CO 2 , 37° C. incubator. After 7 days, the supernatants were collected and purified by high-speed centrifugation, vacuum filtration through a microfiltration membrane and a HiTrap protein A HP column to give antibodies 20G10, 13E5 and 18H7.
  • a chemical defined medium CD medium; containing 1% streptomycin
  • mRNA was extracted from the cell line LT008 cultured in Example 1 according to the method described in the manual of RNAprep pure Cell/Bacteria Kit (Tiangen, Cat. No. DP430).
  • cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR.
  • PCR-amplified products were directly subjected to TA cloning according to the manual of the pEASY-T1 Cloning Kit (Transgen CT101).
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 1 with a length of 348 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 2 with a length of 116 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 9, 10 and 11, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 3 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 4 with a length of 107 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 12, 13 and 14, respectively.
  • variable region sequences of antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 were given by computer modeling and mutation design (antibody constant region sequences from NCBI database: the heavy chain constant region is Ig gamma-4 chain C region, ACCESSION No. P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION No. P01834).
  • variable region sequences are as follows:
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 21 with a length of 348 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 22 with a length of 116 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 9, 10 and 11, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 23 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 24 with a length of 107 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 12, 13 and 14, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 25 with a length of 348 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 26 with a length of 116 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 9, 10 and 11, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 27 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 28 with a length of 107 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 12, 13 and 14, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 29 with a length of 348 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 30 with a length of 116 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 9, 10 and 11, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 31 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 32 with a length of 107 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 12, 13 and 14, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33 with a length of 348 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 34 with a length of 116 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 9, 10 and 11, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 35 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 36 with a length of 107 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 12, 13 and 14, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 33, and
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 27, and
  • the heavy chain constant regions are Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant regions are Ig kappa chain C region, ACCESSION: P01834.
  • the heavy and light chain variable region cDNA of 13E5 H1L1, heavy and light chain variable region cDNA of 13E5 H2L2, heavy and light chain variable region cDNA of 13E5 H3L3, heavy and light chain variable region cDNA of 13E5 H4L2, and heavy and light chain variable region cDNA of 13E5 H4L4 were separately cloned into pUC57 simple vector (provided by Genscript Biotech Corporation) to give pUC57simple-13E5H1, pUC57simple-13E5L1, pUC57simple-13E5H2, pUC57simple-13E5L2, pUC57simple-13E5H3, pUC57simple-13E5L3, pUC57simple-13E5H4 and pUC57simple-13E5L4.
  • variable region fragments were acquired by digestion according to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), and subcloned into the pcDNA3.1 vectors containing the corresponding heavy or light chain constant region fragment (for the pcDNA3.1 vectors containing the heavy and light chain constant regions, the restriction enzymes were HindIII & EcoRI) to give pcDNA3.1-13E5H1, pcDNA3.1-13E5L1, pcDNA3.1-13E5H2, pcDNA3.1-13E5L2, pcDNA3.1-13E5H3, pcDNA3.1-13E5L3, pcDNA3.1-13E5H4 and pcDNA3.1-13E5L4.
  • the recombinant plasmid pair containing the corresponding light and heavy chains (pcDNA3.1-13E5H1 and pcDNA3.1-13E5L1, pcDNA3.1-13E5H2 and pcDNA3.1-13E5L2, pcDNA3.1-13E5H3 and pcDNA3.1-13E5L3, pcDNA3.1-13E5H4 and pcDNA3.1-13E5L4, and pcDNA3.1-13E5H4 and pcDNA3.1-13E5L2) were co-transfected into 293F cells. The cultures were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The cultures were collected after 7 days, and purified on a Protein A column to give humanized antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4.
  • RNAprep pure Cell/Bacteria Kit Tetrachloroetrachloroetrachloride
  • cDNA was synthesized according to the manual of Invitrogen SuperScript® III First-Strand Synthesis System for RT-PCR and amplified by PCR.
  • PCR-amplified products were directly subjected to TA cloning according to the manual of the pEASY-T1 Cloning Kit (Transgen CT101).
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 5 with a length of 360 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 6 with a length of 120 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 15, 16 and 17, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 7 with a length of 333 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 8 with a length of 111 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 18, 19 and 20, respectively.
  • variable region sequences of antibodies 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, 18H7 H3L2 and 18H7 H3L3 were given by computer modeling and mutation design (antibody constant region sequences from NCBI database: the heavy chain constant region is Ig gamma-4 chain C region, ACCESSION No. P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION No. P01834).
  • variable region sequences are as follows:
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 37 with a length of 360 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 38 with a length of 120 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 15, 16 and 17, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 39 with a length of 333 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 40 with a length of 111 amino acids, while the amino acid sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 18, 19 and 20, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 41 with a length of 360 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 42 with a length of 120 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 15, 16 and 17, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 43 with a length of 333 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 44 with a length of 111 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 18, 19 and 20, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 45 with a length of 360 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 46 with a length of 120 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 15, 16 and 17, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 47 with a length of 333 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 48 with a length of 111 amino acids, while the sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 18, 19 and 20, respectively.
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 41, and
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 47, and
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 45, and
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 43, and
  • the heavy chain constant regions are Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant regions are Ig kappa chain C region, ACCESSION: P01834.
  • the heavy and light chain variable region cDNA of 18H7 H1L1, heavy and light chain variable region cDNA of 18H7 H2L2, heavy and light chain variable region cDNA of 18H7 H2L3, heavy and light chain variable region cDNA of 18H7 H3L3, and heavy and light chain variable region cDNA of 18H7 H3L2 were separately cloned into pUC57 simple vector (provided by Genscript Biotech Corporation) to give pUC57simple-18H7H1, pUC57simple-18H7L1, pUC57simple-18H7H2, pUC57simple-18H7L2, pUC57simple-18H7H3 and pUC57simple-18H7L3.
  • variable region fragments were acquired by digestion according to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), and subcloned into the pcDNA3.1 vectors containing the corresponding heavy or light chain constant region fragment to give pcDNA3.1-18H7H1, pcDNA3.1-18H7L1, pcDNA3.1-18H7H2, pcDNA3.1-18H7L2, pcDNA3.1-18H7H3 and pcDNA3.1-18H7L3.
  • the recombinant plasmid pair containing the corresponding light and heavy chains (pcDNA3.1-18H7H1 and pcDNA3.1-18H7L1, pcDNA3.1-18H7H2 and pcDNA3.1-18H7L2, pcDNA3.1-18H7H3 and pcDNA3.1-18H7L3, pcDNA3.1-18H7H2 and pcDNA3.1-18H7L3, and pcDNA3.1-18H7H3 and pcDNA3.1-18H7L2) were co-transfected into 293F cells. The cultures were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression. The cultures were collected after 7 days, and purified on a Protein A column (GE) to give the humanized antibodies 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, 18H7 H3L2 and 18H7 H3L3.
  • GE
  • variable region sequences of antibody 20G10 H3L3 were given by computer modeling and mutation design (antibody constant region sequences from NCBI database: the heavy chain constant region is Ig gamma-4 chain C region, ACCESSION No. P01861.1; the light chain constant region is Ig kappa chain C region, ACCESSION No. P01834).
  • variable region sequences are as follows:
  • the nucleic acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 126 with a length of 360 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 127 with a length of 120 amino acids, while the sequences of heavy chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NO: 130, 131 and 132, respectively.
  • the nucleic acid sequence of the light chain variable region is set forth in SEQ ID NO: 128 with a length of 321 bp.
  • the coded amino acid sequence is set forth in SEQ ID NO: 129 with a length of 107 amino acids, while the amino acid sequences of light chain CDR1, CDR2 and CDR3 are set forth in SEQ ID NOs: 133, 134 and 135, respectively.
  • the heavy chain constant regions are Ig gamma-4 chain C region, ACCESSION: P01861.1; the light chain constant regions are Ig kappa chain C region, ACCESSION: P01834.
  • the heavy and light chain variable region cDNA of 20G10 H3L3 were cloned into pUC57simple vector (provided by Genscript Biotech Corporation) to give pUC57simple-20G10H3 and pUC57simple-20G10L3.
  • the variable region fragments were acquired by digestion according to the standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), and subcloned into the pcDNA3.1 vectors containing the corresponding heavy or light chain constant region fragment to give pcDNA3.1-20G10H3 and pcDNA3.1-20G10L3.
  • the recombinant plasmid pair containing the corresponding light and heavy chains (pcDNA3.1-20G10H3 and pcDNA3.1-20G10L3) were co-transfected into 293F cells.
  • the cultures were collected and purified. After the sequences were verified, endotoxin-free expression plasmids were prepared, and were transiently transfected into HEK293 cells for antibody expression.
  • the cultures were collected after 7 days, and purified on a Protein A column (GE) to give the humanized antibody 20G10 H3L3.
  • GE Protein A column
  • the ELISA plates were coated with human IL4RA-hFc or human IL4RA-mFc and were incubated, and target antibodies were added after blocking. Goat anti-mouse IgG (H+L) and HRP (purchased from Jackson ImmunoResearch Inc., Cat No. 109-035-062), or goat anti-human IgG and HRP (purchased from Jackson ImmunoResearch Inc., Cat No. 109-035-088) were added. The plates were incubated and washed before TMB (Neogen, 308177) was added for chromogenic reaction. At the end of the reaction, the absorbance at 450 nm was measured by a plate reader. The data were analyzed by SoftMax Pro 6.2.1.
  • 13E5, 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 are all capable of binding to antigens human IL4RA-hFc or human IL4RA-mFc effectively, and demonstrate comparable binding activities to that of the reference antibody dupilumab against the same target ( FIGS. 1 , 2 and 3 ).
  • the EC 50 (median effect concentration) of 13E5, 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 for competing with ligand human IL4-N-His for binding to the antigen human IL4RA-hFc was assayed by ELISA, so as to investigate the activity of 13E5, 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2 and 13E5 H4L4 for blocking the binding of the ligand to the target antigen human IL4RA-hFc.
  • the ELISA plates were coated with human IL4RA-hFc and blocked before the antibodies were added.
  • An equal volume of human IL4-N-His (synthesized by Akeso, Inc.) was added, and the mixture was well mixed and incubated.
  • mouse anti-His, HRP purchased from CoWin Biosciences, Cat No. CW0285A was added for incubation.
  • the plates were subjected to another wash. Chromogenic reaction was started by adding TMB (Neogen, 308177) and was then terminated. At the end of the reaction, the OD value at 450 nm wavelength was measured by a plate reader, and was analyzed and processed by SoftMax Pro 6.2.1. The results are shown in Tables 4, 5 and 6.
  • Antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H4L2 and 13E5 H4L4 demonstrate superior activities for competing with human IL4-N-His for binding to human IL4RA-hFc as compared to that of the reference antibody dupilumab for the same antigen.
  • the ELISA plates were coated with human IL4RA-hFc or human IL4RA-mFc and were incubated, and target antibodies were added after blocking. Goat anti-mouse IgG (H+L) and HRP (purchased from Jackson ImmunoResearch Inc., Cat No. 109-035-062), or goat anti-human IgG and HRP (purchased from Jackson ImmunoResearch Inc., Cat No. 109-035-088) were added. The plates were incubated and washed before TMB (Neogen, 308177) was added for chromogenic reaction. At the end of the reaction, the absorbance at 450 nm was measured by a plate reader. The data were analyzed by SoftMax Pro 6.2.1.
  • 18H7, 18H7 H1L1, 18H7 H2L2, 18H7 H2L3, 18H7 H3L2 and 18H7 H3L3 are all capable of binding to antigens human IL4RA-hFc or human IL4RA-mFc effectively, and antibodies 18H7 H1L1, 18H7 H2L2 and 18H7 H2L3 demonstrate comparable binding activities to that of the reference antibody dupilumab against the same target ( FIGS. 7 and 8 ).
  • the EC 50 (median effect concentration) of 18H7, 18H7 H1L1, 18H7 H2L2, 18H7 H2L3 and 18H7 H3L2 for competing with ligand human IL4-N-His for binding to the antigen human IL4RA-hFc was assayed by ELISA, so as to investigate the activity of 18H7, 18H7 H1L1, 18H7 H2L2, 18H7 H2L3 and 18H7 H3L2 for blocking the binding of the ligand to the target antigen human IL4RA-hFc.
  • the ELISA plates were coated with human IL4RA-hFc and blocked before the antibodies were added.
  • An equal volume of human IL4-N-His (synthesized by Akeso, Inc., an IL4 connected with 6 His tags at the N terminus) was added, and the mixture was well mixed and incubated.
  • mouse anti-His, HRP purchased from CoWin Biosciences, Cat No. CW0285M was added for incubation.
  • the plates were subjected to another wash. Chromogenic reaction was started by adding TMB (Neogen, 308177) and was then terminated. At the end of the reaction, the OD value at 450 nm wavelength was measured by a plate reader, and was analyzed and processed by SoftMax Pro 6.2.1. The results are shown in Tables 9 and 10.
  • 18H7, 18H7 H1L1, 18H7 H2L2 and 18H7 H3L2 are all capable of effectively blocking the binding of the ligand human IL4-N-His to the antigen human IL4RA-hFc in a dose-dependent manner.
  • Antibodies 18H7 H1L1, 18H7 H2L2 and 18H7 H3L2 demonstrate superior activities for competing with human IL4-N-His for binding to human IL4RA-hFc as compared to that of the reference antibody dupilumab for the same antigen.
  • the EC 50 (median effect concentration) of 20G10 H3L3 and 13E5 H4L4 for competing with ligand human IL4-N-His for binding to the antigen human IL4RA-hFc was assayed by ELISA, so as to investigate the activity of 20G10 H3L3 and 13E5 H4L4 for blocking the binding of the ligand to the target antigen human IL4RA-hFc.
  • the ELISA plates were coated with human IL4RA-hFc and blocked before the antibodies were added.
  • An equal volume of human IL4-N-His (synthesized by Akeso, Inc.) was added, and the mixture was well mixed and incubated.
  • mouse anti-His, HRP purchased from CoWin Biosciences, Cat No. CW0285A was added for incubation.
  • the plates were subjected to another wash. Chromogenic reaction was started by adding TMB (Neogen, 308177) and was then terminated. At the end of the reaction, the OD value at 450 nm wavelength was measured by a plate reader, and was analyzed and processed by SoftMax Pro 6.2.1. The results are shown in Table 11.
  • 20G10 H3L3 and 13E5 H4L4 are all capable of effectively blocking the binding of the ligand human IL4-N-His to the antigen human IL4RA-hFc in a dose-dependent manner.
  • the antibodies 20G10 H3L3 and 13E5 H4L4 demonstrate superior activities for competing with human IL4-N-His for binding to human IL4RA-hFc as compared to that of the reference antibody dupilumab for the same antigen.
  • Example 8 Affinity Constant Assay of Humanized Antibodies 13E5 H1L1, 13E5 H2L2, 13E5 H3L3, 13E5 H4L2, 13E5 H4L4, 18H7 H1L1, 18H7 H2L2 and 18H7 H3L2 for Human IL-4RA
  • association rate constants of 13E5 H1L1 and 13E5 H4L2 with the antigen human IL-4RA are greater than that of the reference antibody dupilumab, suggesting faster binding of 13E5 H1L1 and 13E5 H4L2 to the antigen human IL-4RA than dupilumab.
  • TF-1 cells purchased from American Type Culture Collection, Cat. No. CRL-2003 were cultivated (complete medium: RPMI 1640 + 10% FBS + 2.5 g/L glucose + 2 ng/mL GM-CSF). On the day of assay, TF-1 cells were separated by centrifugation, resuspended in a medium free of GM-CSF, and counted. The cells were inoculated into 96-well plates at 20,000 cells/well.
  • the treatment was given as designed: the final concentrations of the antibodies were 0.05, 0.5 and 5 nM; three concentrations 0.041, 0.41 and 4.1 nM were set for IL-4, and the antibody group employed 0.41 nM IL-4; three concentrations 1.58, 15.8 and 79 nM were set for IL-13, and the antibody panel employed 15.8 nM IL-13.
  • the 96-well plates were incubated in a 5% carbon dioxide incubator at 37° C. for 72 h. After the 72 hours, CCK8 reagent was added according to the instruction of CCK8 kit (purchased from Dojindo Laboratories, Japan, Cat No. CK04). The mixture was mixed well, incubated at 37° C.
  • a curve of OD vs. cell number was plotted by seeding serially diluted TF-1 cells into 96-well plates, adding CCK8 reagent, incubating the plates in a 5% carbon dioxide incubator at 37° C. for 3-4 h and reading the OD value at 450 nm.
  • the cell numbers of the groups were calculated according to the OD value, and GraphPad Prism 5 were used for plotting.
  • the isotype reference antibody was human anti-hen egg lysozyme IgG (anti-HEL) derived from the variable region of the Fab F10.6.6 sequence in Affinity Maturation Increases the Stability and Plasticity of the Fv Domain of Anti-Protein Antibodies (Acierno et al., J Mol Biol. , 2007, 374(1):130-46).
  • the isotype was synthesized by Akeso, Inc.
  • Nanjing Genscript Biology was entrusted for codon optimization and gene synthesis for the heavy and light chain (complete sequence or variable region) genes of the human IgG antibodies.
  • the heavy and light chain genes were subcloned into the heavy and antibody light chain expression vectors (both were pcDNA3.1 vector) for mammalian expression system by standard molecular cloning techniques such as PCR, enzymatic digestion, DNA gel electrophoresis, ligation and transformation, colony PCR or digestion and identification.
  • the heavy and light chain genes of the recombinant expression vector were further sequenced and analyzed (the heavy and light chain sequences are set forth in SEQ ID NOs: 122 and 123).
  • endotoxin-free expression plasmids were prepared in a large scale, and the heavy and light chain plasmids were transiently co-transfected into HEK293 cells for expression of recombinant antibody. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column (GE), and the quality of the resulting antibody sample was determined using SDS-PAGE and SEC-HPLC standard analysis techniques.
  • GE Protein A column
  • both human IL-4 and human IL-13 are able to effectively promote the proliferation of TF-1 cells in a dose-dependent manner; as compared to isotype reference antibodies (human IgG), dupilumab, 13E5 H1L1, 13E5 H2L2, 13E5 H4L2, 13E5 H4L4, 18H7 H1L1 and 18H7 H2L2 can specifically inhibit TF-1 cell proliferation induced by IL-4 and IL-13 in a dose-dependent manner.
  • Normal human peripheral blood (with heparin) was separated by Ficoll density gradient centrifugation to give human fresh PBMCs. After 3 centrifugations and washes, the cells were counted and the density was adjusted to 2.5 ⁇ 10 6 cells/mL. The cells were seeded into low attachment 96-well plates at 200 ⁇ L of PBMCs per well (i.e., 500,000 cells/well); 25 ⁇ L of the antibodies dupilumab, 13E5 H4L4 or 18H7 H1L1 (at final concentrations of 30, 3 and 0.3 nM, respectively) were added to each well, and blank reference and isotype reference human IgG (at a final concentration of 30 nM) were set.
  • the cells were incubated at room temperature for 30 min. After 30 min, 25 ⁇ L of human IL-4 (final concentration of 100 pM) or 25 ⁇ L of human IL-13 (final concentration of 300 ng/mL) was added and the system was incubated for 2.5 days. After the 2.5 days, the PBMCs were separated and transferred into 1.5-mL EP tubes, added with 500 ⁇ L of 1% PBSA and centrifuged at 1000 ⁇ g for 5 min. The supernatant was discarded, and the residues were added with 50 ⁇ L of CD23-PE antibody (50-fold diluted with 1% PBSA) and incubated on ice for 40 min.
  • the system was added with 1 mL of 1% PBSA and centrifuged at 1000 ⁇ g for 5 min before the supernatant was discarded.
  • the cells were resuspended in 200 ⁇ L of 1% PBSA, and transferred into a flow cytometry tube for assay.
  • a B-hIL4Ra mouse (C57BL/6 background) skin inflammation model was established by inducing with HDM (human dust mite allergen, Greer Laborct, Lot No. 348717).
  • the mice were divided into 6 groups with 7 mice in each, including isotype reference group (anti-HEL), dupilumab high-dose group (80 mg/kg; commercially available dupilumab from Sanofi and Regeneron Pharmaceuticals) and low-dose group (20 mg/kg; commercially available as Dupilumab from Sanofi and Regeneron Pharmaceuticals), 13E5 H4L4 high-dose group (80 mg/kg) and low-dose group (20 mg/kg).
  • the treatment was given by subcutaneous injection on D-1, D2, D5 and D8.
  • the animal model was established by daily intradermal injection of normal saline.
  • the animal model was established with daily intradermal injection of HDM (37.5 ⁇ g ⁇ 2/25 ⁇ L/day) for 7 days and daily intradermal injection of HDM (25 ⁇ g ⁇ 2/25 ⁇ L/day)for 3 days.
  • the animal model was established with daily intradermal injection of HDM (37.5 ⁇ g ⁇ 2/25 ⁇ L/day)for 7 days and daily intradermal injection of HDM (25 ⁇ g ⁇ 2/25 ⁇ L/day)for 3 days.
  • Epidermal thickness of the mice was measured to investigate the effect of 13E5 H4L4 in inhibiting the epidermal thickness in B-hIL4Ra mouse skin inflammation model.
  • D0 is the first day of model establishment.
  • the normal saline and HDM were administered at the upper right back and lower right back respectively.
  • the results are shown in FIG. 30 .
  • 13E5 H4L4 of different doses can effectively inhibit the thickening of epidermis, and the blood concentration of 13E5 H4L4 is higher than that of dupilumab at 80 mg/kg, which corresponds to the efficacy.
  • the pathological sections are shown in FIG. 31 : in the B-hIL4Ra mouse skin inflammation model established by 37.5 ⁇ g + 25 ⁇ g HDM intradermal injection, the thickness of epidermis in the model group is significantly thickened as compared with that of the normal group, the thickening of epidermis in mice can be significantly inhibited by dupilumab 80 mg/kg and 13E5 H4L4 80 mg/kg subcutaneous injections with good dose-response relationship. 13E5 H4L4 80 mg/kg is slightly superior to dupilumab 80 mg/kg.
  • HCDR1 GYTFTEYT (SEQ ID NO: 9)
  • HCDR2 INPNNGGT (SEQ ID NO: 10)
  • HCDR3 ARVRRGMDY (SEQ ID NO: 11)
  • LCDR1 QDVTTA (SEQ ID NO: 12)
  • LCDR3 QQHYSAPWT (SEQ ID NO: 14)
  • HCDR1 GFTFSSSY (SEQ ID NO: 15)
  • HCDR2 INSNGGKT (SEQ ID NO: 16)
  • HCDR3 TRQRGNYVGAMDY (SEQ ID NO: 17)
  • LCDR1 QDVSTA (SEQ ID NO: 18)
  • LCDR3 HQYYGSPPT (SEQ ID NO: 20)
  • FR-H1 EVQLQQSGPELVKPGASVKISCKTS (SEQ ID NO: 49)
  • FR-H3 VYNQNFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYC (SEQ ID NO: 51)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 52)
  • FR-L1 DIVMTQSHKFMSTSVGDRVSITCKAS (SEQ ID NO: 53)
  • FR-L2 VAWYQQKPGQSPKLLIY (SEQ ID NO: 54)
  • FR-L3 YRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC (SEQ I D NO: 55)
  • FR-L4 FGGGTNLEIK (SEQ ID NO: 56)
  • FR-H1 DVKLVESGGDLVNLGGSLKLSCAAS (SEQ ID NO: 57)
  • FR-H3 YYPDTVKGRFTISRDNAKNTLYLQMSGLKTEDTALYYC (SEQ ID NO: 59)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 60)
  • FR-L1 DIVMTQSHKFMSTSVGDRVSITCKAS (SEQ ID NO: 61)
  • FR-L2 VVWYQQKPGQSPTLLIY (SEQ ID NO:62)
  • FR-L3 YRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYC (SEQ I D NO: 63)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 64)
  • FR-H2 IHWVKQAHGQSLEWIGG (SEQ ID NO: 66)
  • FR-H3 VYNQKFQGKATLTVDKSTSTAYMELRSLTSEDTAVYYC (SEQ ID NO: 67)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 68)
  • FR-L1 DIQMTQSPKSLSTSVGDRVTITCRAS (SEQ ID NO: 69)
  • FR-L2 VAWYQQKPGKSPKLLIY (SEQ ID NO: 70)
  • FR-L3 YRYTGVPSRFSGSGSGTDFTFTISSVQPEDLATYYC (SEQ I D NO: 71)
  • FR-L4 FGGGTNLEIK (SEQ ID NO: 72)
  • FR-H1 QVQLVQSGAEVVKPGASVKVSCKTS (SEQ ID NO: 73)
  • FR-H2 IHWVRQAPGQSLEWIGG (SEQ ID NO: 74)
  • FR-H3 NYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 75)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 76)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 77)
  • FR-L2 VAWYQQKPGKAPKLLIY (SEQ ID NO: 78)
  • FR-L3 SRYTGVPSRFSGSGSGTDFTLTISSVQPEDLATYYC (SEQ I D NO: 79)
  • FR-L4 FGGGTNLEIK (SEQ ID NO: 80)
  • FR-H1 QVQLVQSGAEVVKPGASVKVSCKAS (SEQ ID NO: 81)
  • FR-H2 IHWVRQAPGQGLEWIGG (SEQ ID NO: 82)
  • FR-H3 NYAQKFQGRVTITVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 83)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 84)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 85)
  • FR-L2 LAWYQQKPGKAPKLLIY (SEQ ID NO: 86)
  • FR-L3 SLYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ I D NO: 87)
  • FR-L4 FGGGTNLEIK (SEQ ID NO: 88)
  • FR-H1 QVQLVQSGAEVVKPGASVKVSCKTS (SEQ ID NO: 89)
  • FR-H3 VYNQKFQGKVTLTVDKSTSTAYMELSSLRSEDTAVYYC (SEQ ID NO: 91)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 92)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 93)
  • FR-L2 VAWYQQKPGKAPKLLIY (SEQ ID NO: 94)
  • FR-L3 YRYTGVPSRFSGSGSGTDFTLTISSVQPEDLATYYC (SEQ I D NO: 95)
  • FR-L4 FGGGTNLEIK (SEQ ID NO: 96)
  • FR-H1 DVQLVESGGGLVQLGGSLRLSCAAS (SEQ ID NO: 97)
  • FR-H3 YYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYC (SEQ ID NO: 99)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 100)
  • FR-L1 DIQMTQSPSSLSTSVGDRVTITCKAS (SEQ ID NO: 101)
  • FR-L2 VVWYQQKPGKSPKLLIY (SEQ ID NO: 102)
  • FR-L3 YRYTGVPSRFSGSGSGTDFTFTISSVQPEDIATYYC (SEQ I D NO: 103)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 104)
  • FR-H1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 105)
  • FR-H3 YYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYC (SEQ ID NO: 107)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 108)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCQAS (SEQ ID NO: 109)
  • FR-L2 VVWYQQKPGKSPKLLIY (SEQ ID NO: 110)
  • FR-L3 YLYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ I D NO: 111)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 112)
  • FR-H1 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 113)
  • FR-H3 YYADSVKGRFTISRDNSKNTLYLQMSSLRAEDTAVYYC (SEQ ID NO: 115)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 116)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCQAS (SEQ ID NO: 117)
  • FR-L2 LAWYQQKPGKAPKLLIY (SEQ ID NO: 118)
  • FR-L3 NLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ I D NO: 119)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 120)
  • HCDR1 GFSLSTSGMG (SEQ ID NO: 130)
  • HCDR2 IWWADDK (SEQ ID NO: 131)
  • HCDR3 ARITRGNSAMDF (SEQ ID NO: 132)
  • LCDR1 ENVYSY (SEQ ID NO: 133)
  • LCDR2 NAK (SEQ ID NO: 134)
  • LCDR3 QHHYGIPWT (SEQ ID NO: 135)
  • FR-H1 QVTLKESGPTLVKPTQTLTLTCTFS (SEQ ID NO: 136)
  • FR-H2 VGWIRQPPGKALEWLAL (SEQ ID NO: 137)
  • FR-H3 RYSTSLKSRLTISKDTSKNQVVLTITNVDPVDTATYYC (SEQ ID NO: 138)
  • FR-H4 WGQGTSVTVSS (SEQ ID NO: 139)
  • FR-L1 DIQMTQSPSSLSASVGDRVTITCRAS (SEQ ID NO: 140)
  • FR-L2 LNWYQQKPGKAPKLLIY (SEQ ID NO: 141)
  • FR-L3 SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ I D NO: 142)
  • FR-L4 FGGGTKLEIK (SEQ ID NO: 143)

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CN113101364B (zh) * 2018-05-29 2023-12-01 康诺亚生物医药科技（成都）有限公司 一种自免疫抑制剂的开发和应用
CN113527485A (zh) 2020-04-17 2021-10-22 上海麦济生物技术有限公司 抗人白细胞介素-4受体α抗体及其制备方法和应用
CN114555639B (zh) * 2020-09-10 2023-12-12 舒泰神（北京）生物制药股份有限公司 特异性识别白细胞介素-4受体α的抗体及其用途
CN114457024A (zh) * 2020-10-30 2022-05-10 未来智人再生医学研究院(广州)有限公司 一种表达IL-4Rα阻断物的多能干细胞或其衍生物及应用
CN114634574B (zh) * 2022-04-18 2022-12-20 先进生物(苏州)有限公司 抗B7H6的scFv抗体、其编码基因及其应用
CN117085122A (zh) * 2022-04-29 2023-11-21 中山康方生物医药有限公司 抗人il-4ra的抗体及其用途
TW202406572A (zh) 2022-05-02 2024-02-16 美商再生元醫藥公司 抗介白素－４受體（ｉｌ－４ｒ）抗體調配物
WO2023215750A2 (fr) 2022-05-02 2023-11-09 Regeneron Pharmaceuticals, Inc. Procédés de réduction de l'activité de lipase

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CA2708871C (fr) * 2007-12-21 2017-11-21 Medimmune Limited Elements liants destines au recepteur alpha de l'interleukin-4(il-4r-.alpha.) - 173
PE20190658A1 (es) * 2012-02-24 2019-05-08 Abbvie Stemcentrx Llc Moduladores y metodos de empleo novedosos
US9574004B2 (en) * 2012-08-21 2017-02-21 Sanofi Biotechnology Methods for treating or preventing asthma by administering an IL-4R antagonist
TWI633891B (zh) * 2013-06-04 2018-09-01 再生元醫藥公司 藉由投與ｉｌ－４ｒ抑制劑以治療過敏及增強過敏原－特異之免疫療法的方法
TWI634900B (zh) * 2013-07-11 2018-09-11 再生元醫藥公司 藉由投與ｉｌ－４ｒ抑制劑治療嗜酸性食道炎的方法
US8986691B1 (en) * 2014-07-15 2015-03-24 Kymab Limited Method of treating atopic dermatitis or asthma using antibody to IL4RA
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WO2016034648A1 (fr) * 2014-09-03 2016-03-10 Medimmune Limited Formulation stable d'anticorps anti-il-4r-alpha
PT3973987T (pt) * 2014-09-15 2024-04-01 Regeneron Pharma Combinação compreendendo um antagonista de il-4r, um corticoesteroide e um agonista beta2-adrenérgico de ação prolongada para tratar a asma
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CN108373505B (zh) * 2018-04-20 2019-08-20 北京智仁美博生物科技有限公司 抗il-4r抗体及其用途

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BR112021012647A2 (pt) 2021-09-14
EP3904385A4 (fr) 2023-04-19
AU2019412405A1 (en) 2021-09-02
JP2022519340A (ja) 2022-03-23
CN111647082A (zh) 2020-09-11
CN116813770A (zh) 2023-09-29
EP3904385A1 (fr) 2021-11-03
EA202191806A1 (ru) 2021-11-09
PH12021551534A1 (en) 2022-02-28
JP2023126929A (ja) 2023-09-12
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